Efficacy and safety of recombinant human granulocyte colony-stimulating factor in patients with unexplained recurrent pregnancy loss: a protocol for a multicenter, randomized, double-blind, controlled trial.

Access to data will be limited to qualified personnel with unique password-protected accounts. The Independent Data and Safety Monitoring Board (IDSMB) monitors data impacting the safety of patients, provides independent expertise for the assessment of AEs occurring during the study, and comments and recommendations on the benefit-risk ratio to assist the Steering Committee in making decisions regarding protocol modifications or early termination of the study, regularly assesses the progress of the clinical trial, safety data, and primary efficacy endpoints, and recommends to the sponsor on whether to continue, modify, or discontinue the trial to ensure the interests and health of the subjects. IDSMB consists of eight experts who have no conflict of interest with this study, are not directly involved in the study, and are independent of the sponsor. Six members with voting rights include two obstetrician-gynecologists, two clinical pharmacologists, and two clinical research methodologists, while two independent statisticians have no voting rights. IDSMB will forward its recommendation to the Steering Committee, which will decide whether to stop the study. Taking safety data and efficacy into account, IDSMB can offer several different recommendations: (1) to continue the study without modifying the protocol; (2) to continue the study with modifying the protocol; (3) to temporarily stop the enrollment; and (4) to early terminate the study. It refers to any untoward medical event that occurs from the time the subject signs the informed consent form and is enrolled in the trial to the last follow-up, which will be judged as an adverse event regardless of its causal relationship with the investigational drug. It refers to any clinical event indicating significant harm, contraindications, side effects, or requiring caution. An adverse event is classified as serious if it meets one or more of the following criteria: death; life-threatening (defined as an event of which the patient is at immediate risk of death at the time of the event, not including events that, if more severe, may cause the patient’s death); resulting in hospitalization or prolongation of hospitalization; and resulting in persistent or significant loss of labor or disability. Some medical events that have not resulted in death, life-threatening events, or required hospitalization should also be considered as SAEs when, based on appropriate medical judgment, they may jeopardize the patient or the subject or may require medical or surgical treatment to avoid the above. It refers to any adverse event and hematological and/or other laboratory abnormalities that lead to targeted medical measures (such as drug withdrawal, dose reduction and symptomatic treatment) in addition to serious adverse events. All clinical adverse events occurring in this clinical study will be documented on the case report form (CRF) Adverse Event page. Adverse event intensity will be graded. For uniform criteria, the intensity of the event is graded as follows: u Mild: Perceptible discomfort without interfering with daily activities. u Moderate: High discomfort causing interference with or reduction in daily activities. u Severe: Inability to work or daily activities. Mild: Perceptible discomfort without interfering with daily activities. Moderate: High discomfort causing interference with or reduction in daily activities. Severe: Inability to work or daily activities. Criteria for judging the relationship between adverse events and investigational drug. Causal analysis of the relationship between all adverse events and the investigational drug will be judged according to five levels: definitely related, probably related, possibly related, possibly unrelated, and definitely unrelated, and the first three levels are defined as adverse drug reactions. Causal analysis considerations include the following five aspects: u Whether there is a reasonable sequential relationship between the start time of medication and the occurrence time of suspected adverse drug reaction (ADR) (occurrence after medication) u Whether the suspected ADR is consistent with the known ADR of the drug (consistent with literature). u Whether the suspected ADR can be explained by concomitant medication, previous medication, the patient’s clinical condition, or the influence of other therapies (other explanations). u Whether the suspected ADR disappears or abates after drug withdrawal or dose reduction (drug withdrawal reactions). u Whether the suspected ADR recurs after re-exposure to the same drug (recurrence after re-medication). Whether there is a reasonable sequential relationship between the start time of medication and the occurrence time of suspected adverse drug reaction (ADR) (occurrence after medication) Whether the suspected ADR is consistent with the known ADR of the drug (consistent with literature). Whether the suspected ADR can be explained by concomitant medication, previous medication, the patient’s clinical condition, or the influence of other therapies (other explanations). Whether the suspected ADR disappears or abates after drug withdrawal or dose reduction (drug withdrawal reactions). Whether the suspected ADR recurs after re-exposure to the same drug (recurrence after re-medication). The investigator should evaluate the possible relationship between adverse events and the investigational drug with concomitant drugs, with reference to the following table (Table  1 ). Table 1 The relationship between adverse events and the investigational drug with concomitant drugs Considerations Occurrence after medication Consistent with literature Other explanations Disappearance after drug withdrawal Recurrence after re-medication Definitely related  +   +   −   +   +  Probably related  +   +   −   +  ? Possibly related  +   +   ±   ±  ? Possibly unrelated  +   −   ±   ±  ? Definitely unrelated  −   −   +   −   −  Note: “ + ” = yes; “ − ” = no; “ ± ” = possibly; “?” = uncertain The relationship between adverse events and the investigational drug with concomitant drugs Note: “ + ” = yes; “ − ” = no; “ ± ” = possibly; “?” = uncertain The project management group will hold regular meetings, typically on a monthly basis, to review the overall progress of the trial. These meetings will include discussions on participant recruitment status (compared to projected targets), data completeness and quality, retention rates, protocol compliance, and any operational or ethical issues arising during implementation. Additionally, the coordinating team will prepare progress reports at the midpoint and upon completion of the study. These reports will be submitted to the local ethics committees and will summarize recruitment outcomes, monitoring activities, encountered challenges, and any deviations from the approved protocol. Any protocol amendments will first be submitted to the sponsor and funder for approval. Once approved, the principal investigator will notify all participating centers and provide updated versions of the protocol for inclusion in the Investigator Site File. All deviations from the protocol will be documented using breach report forms. The trial registry entry will also be updated accordingly. The findings of this trial will be disseminated through publication in peer-reviewed scientific journals and presentations at national and international conferences relevant to reproductive medicine. The results will also be shared with participating study sites and local ethics committees. Efforts will be made to publish the results in an open-access format to ensure broad accessibility.

The study will take place at Lanzhou University Second Hospital (Lanzhou, China), the First Affiliated Hospital of Chongqing Medical University (Chongqing, China), the Third Affiliated Hospital of Wenzhou Medical University (Wenzhou, China), and Hebei Reproductive Health Hospital (Shijiazhuang, China). The study population includes women with the loss of at least two pregnancies, excluding those with known pathogenesis, and willingness to conceive spontaneously. u Age of 20 to 37 years old (the reason for setting the upper age limit of 37 years old is that abortion caused by chromosomal aberrations has a higher incidence in advanced maternal age and cannot be prevented by immunological regulation). u Body mass index (BMI) between 15 and 29 at screening. u Menstrual cycle within 21–35 days, and the difference between the longest cycle and the shortest cycle only in 1 year is no more than 7 days. u History of recurrent pregnancy loss, defined as two or more fetal losses before 24 weeks of gestation (including biochemical pregnancy). u Willingness to conceive spontaneously and signing of informed consent form. Age of 20 to 37 years old (the reason for setting the upper age limit of 37 years old is that abortion caused by chromosomal aberrations has a higher incidence in advanced maternal age and cannot be prevented by immunological regulation). Body mass index (BMI) between 15 and 29 at screening. Menstrual cycle within 21–35 days, and the difference between the longest cycle and the shortest cycle only in 1 year is no more than 7 days. History of recurrent pregnancy loss, defined as two or more fetal losses before 24 weeks of gestation (including biochemical pregnancy). Willingness to conceive spontaneously and signing of informed consent form. u Gestational age > 5 weeks + 6 days. u Chromosomal abnormalities in females or their partners, including numerical and structural aberrations. u Patients previously diagnosed with infertility (no conception after regular sexual life without any contraception for at least 1 year). u Exclusion of immunological factors including autoimmune disease (e.g., antiphospholipid syndrome, systemic lupus erythematosus, undifferentiated connective tissue disease, Sjogren’s syndrome, rheumatoid arthritis and systemic sclerosis); thrombophilia (anticoagulant protein C, S deficiency, antiphospholipid syndrome and hyperhomocysteinemia, etc.); anatomical factors including congenital anatomical abnormalities (mediastinal uterus, bicornuate uterus, arcuate uterus, unicornuate uterus, duplex uterus, uterine dysplasia and congenital cervical insufficiency, etc.) and acquired anatomical abnormalities (Asherman syndrome, cervical insufficiency, adenomyosis, uterine fibroids, and endometriosis, etc.); endocrine factors (polycystic ovary syndrome (PCOS), luteal phase dysfunction, hyperprolactinemia (HPRL), thyroid disease requiring drug treatment, diabetes, etc.) and other medical history. u Combination of the following diseases with uncontrolled symptoms: asthma, hypertension, infection. u Present with the following laboratory abnormalities at screening or within the first 3 months: platelet decreased or increased (platelet count  500,000/μL); neutrophil decreased or increased (absolute neutrophil count  10,000/μL), white blood cell decreased or increased (white blood cell count  15,000/μL), and creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, or uric acid > 1.5 times the upper limit of normal. u Allergy to G-CSF or any component of the injection. u History of G-CSF use within the past 3 months. u Other contraindications to the use of investigational drug. Gestational age > 5 weeks + 6 days. Chromosomal abnormalities in females or their partners, including numerical and structural aberrations. Patients previously diagnosed with infertility (no conception after regular sexual life without any contraception for at least 1 year). Exclusion of immunological factors including autoimmune disease (e.g., antiphospholipid syndrome, systemic lupus erythematosus, undifferentiated connective tissue disease, Sjogren’s syndrome, rheumatoid arthritis and systemic sclerosis); thrombophilia (anticoagulant protein C, S deficiency, antiphospholipid syndrome and hyperhomocysteinemia, etc.); anatomical factors including congenital anatomical abnormalities (mediastinal uterus, bicornuate uterus, arcuate uterus, unicornuate uterus, duplex uterus, uterine dysplasia and congenital cervical insufficiency, etc.) and acquired anatomical abnormalities (Asherman syndrome, cervical insufficiency, adenomyosis, uterine fibroids, and endometriosis, etc.); endocrine factors (polycystic ovary syndrome (PCOS), luteal phase dysfunction, hyperprolactinemia (HPRL), thyroid disease requiring drug treatment, diabetes, etc.) and other medical history. Combination of the following diseases with uncontrolled symptoms: asthma, hypertension, infection. Present with the following laboratory abnormalities at screening or within the first 3 months: platelet decreased or increased (platelet count  500,000/μL); neutrophil decreased or increased (absolute neutrophil count  10,000/μL), white blood cell decreased or increased (white blood cell count  15,000/μL), and creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, or uric acid > 1.5 times the upper limit of normal. Allergy to G-CSF or any component of the injection. History of G-CSF use within the past 3 months. Other contraindications to the use of investigational drug. The obstetricians or reproductive medicine physicians participating in this trial from the sites are trained on the knowledge of recombinant human G-CSF (rhG-CSF) for treatment of uRPL by means of online training sessions and on-site training. All departmental personnel participating in this trial from the sites are required to refer potentially eligible women to the G-CSF project leader at each site. Each site has a project leader to ensure the progress of patient recruitment and professional consultation about uRPL at the site. In addition to this recruitment method, recruitment posters are placed at each site to reach more patients. During the screening visit, trial staff will assess patients in strict accordance with the inclusion and exclusion criteria. Informed consent will be obtained from all participants by trained clinicians or trial staff at each participating center prior to enrollment. Eligible women will be identified during routine outpatient visits and provided with a verbal and written explanation of the study objectives, procedures, potential risks, and benefits. They will be given sufficient time to consider participation and to ask questions. Written informed consent will be obtained before any study-specific procedures are conducted. In addition to data and biological samples collected for this study’s primary and secondary outcomes, study participants will be asked to provide optional consent for the storage and future use of anonymized samples and data for related reproductive or immunological research. All stored specimens will be handled in de-identified form and used strictly for ethically approved research purposes. A placebo comparator was chosen to allow for a rigorous assessment of the efficacy and safety of rhG-CSF in women with uRPL. Both the intervention and control groups will receive standard supportive treatment (dydrogesterone and aspirin). This design allows us to isolate the effect of G-CSF while maintaining patient safety and alignment with routine clinical care. The enrolled eligible participants will be randomized into the test group and control group. The test group will receive a subcutaneous injection of 150 μg rhG-CSF (Trade Name: Topneuter; Xiamen Amoytop Biotech Co., Ltd., China) every other day, and the placebo group will receive a subcutaneous injection of the same dose of placebo, starting from the positive pregnancy test to 10 weeks + 0 days of gestation. The dosing strategy of 150 μg rhG-CSF every other day was chosen based on pharmacodynamic data from a previous dose-escalation study, which demonstrated that this regimen maintained stable neutrophil levels in healthy individuals [ 25 ]. The basic medication regimen is oral administration of dydrogesterone and aspirin. Dydrogesterone and aspirin are used empirically in many Chinese fertility clinics to support early pregnancy, particularly in patients with a history of pregnancy loss [ 26 ][ 27 ] [ 28 ]. Although not universally accepted as standard, they are considered part of supportive therapy in clinical practice. Participants may discontinue or modify the allocated intervention under the following conditions: (1) occurrence of any serious adverse event deemed related to the study drug; (2) development of significant medical conditions during the treatment period that, in the investigator’s judgment, make continued administration unsafe (e.g., allergic reaction, severe infection, or abnormal laboratory results); (3) voluntary withdrawal of consent by the participant at any point; (4) investigator or sponsor decision due to concerns about participant safety or protocol compliance. All discontinuations and reasons will be recorded in the case report form. Participants who discontinue the intervention will be encouraged to continue study follow-up for outcome data collection whenever feasible. To promote adherence to the allocated interventions, participants will be provided with both verbal and written instructions detailing the timing and method of administering the subcutaneous injections and oral medications. During the intervention period, trial staff will maintain regular contact with participants through scheduled phone calls to remind them of upcoming doses, reinforce compliance, and offer support for any questions or concerns. Participants will be provided with a medication diary to record each administered dose and will also be encouraged to reach out to the research team at any point if they experience side effects or difficulties following the prescribed regimen. The use of additional immunomodulatory agents, such as corticosteroids, IVIG, or other immunosuppressive therapies, is not permitted during the study, as these may confound the effects of the investigational intervention. Routine prenatal care and non-conflicting medications deemed necessary by the treating clinician, like vitamins, folic acid, and antibiotics, will be allowed, provided they are documented. Any use of prohibited medications or interventions during the trial will be considered a protocol deviation, and they will be excluded from the study. Participants will receive routine obstetrical care after the intervention phase. No additional ancillary or long-term care has been planned. Any participant who experiences an adverse event related to the intervention will receive appropriate medical care at the study site, in accordance with standard clinical protocols. Compensation for injury or harm resulting from trial participation will be provided in accordance with applicable Chinese laws and institutional policies. Participants will also be informed that they may contact the study team at any time after the trial if they have health concerns potentially related to the study. The primary outcome is fetal survival to 24 weeks of gestation. The secondary efficacy outcomes are early pregnancy loss (pregnancy loss occurring before 12 weeks), clinical pregnancy to 10 weeks (presence of gestational sac in utero and fetus with heartbeats confirmed by ultrasound and pregnancy continued to 10 weeks of gestation), ongoing pregnancy to 12 weeks (pregnancy continued to 12 weeks of gestation), fetal survival to 28 weeks, ectopic pregnancy (gestational sac located outside the uterine cavity confirmed by ultrasound), preterm birth (birth at 28 weeks but less than 37 weeks), stillbirth (fetal death during delivery after 28 weeks of gestation), and pregnancy complications (such as preeclampsia, gestational diabetes mellitus [GDM], HELLP syndrome, placental abruption). Safety indicators include (1) maternal adverse reactions, such as musculoskeletal pain, loss of appetite, fever, headache, fatigue, and rash; and (2) laboratory parameters, including white blood cell (WBC) count, neutrophil count or percentage, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), serum creatinine (Cr), blood urea nitrogen (BUN). Postpartum follow-up is conducted to collect neonatal outcomes, including intrauterine growth restriction (IUGR, defined as estimated fetal weight below the 10th percentile for gestational age), gestational age, birth weight, Apgar scores at 1 and 5 min, congenital anomalies confirmed postnatally, neonatal intensive care unit (NICU) admission, and early neonatal death (within 7 days of life). Figure  2 illustrates the complete timeline of treatment administration and outcome assessments across the study period. When contacting potential subjects for the first time, after introducing the trial’s background, objective, and procedures to patients, the trial staff need to conduct a qualification assessment according to the preliminary screening criteria, and will perform baseline visits to the patients who meet the preliminary screening criteria and sign the informed consent form, complete the patient’s medical history collection (physical examination, demographic characteristics, menstrual history, marriage and childbearing history, past disease history, family history, surgical history, drug use, allergy history and living habits) and laboratory test data. The patients with a positive pregnancy test within 3 months shall immediately contact the staff to re-check their baseline data (medical history data, laboratory tests); patients with complete and correct data and meeting the inclusion and exclusion criteria of the trial will be randomized on the same day. After receiving the medicine, the rhG-CSF injection method and medication rules will be taught by a specially assigned person, and the date of the next follow-up will be informed. For patients who have been trying to conceive for more than 3 months, the staff shall ask whether they are still willing to participate in the trial, and those who did need to reassess their baseline data (medical history data, laboratory tests) and update their information. Enrolled patients will be randomized into the test group or placebo group on the day of a positive pregnancy test and start to inject one vial of rhG-CSF or placebo every other day until 10 weeks + 0 days of gestation. During rhG-CSF administration, blood routine (white blood cell count, neutrophil count, etc.) and liver and kidney function tests will be monitored every 2 weeks. The collected blood samples will be uniformly sent to Chongqing KingMed Diagnostics Co., Ltd. by personnel not involved in the trial for testing, and all personnel involved in the trial will not be able to view the blood routine data of the patients. Routine examinations such as three items of pregnancy and ultrasound during pregnancy will be performed according to the requirements of each site and the actual conditions of the patients, and the follow-up personnel will strictly record the examination indicators. After drug withdrawal, the patients will be examined according to the routine obstetrical diagnosis and treatment process, and the follow-up personnel will record the pregnancy status of the patients, part of the examination indicators during the period, pregnancy complications, concomitant medication, and adverse events during the period, pregnancy outcomes, and neonatal conditions in detail according to the time nodes. This study is a randomized controlled trial, with two groups: rhG-CSF group and placebo group, with live birth rate of pregnancy as the main outcome measure. According to the previous literature results (16, 17, 22), it is estimated that the live birth rate is 75% in the rhG-CSF group and 60% in the placebo group, test level α  = 0.05 (two-sided), power 1- β is 90%, sample size distribution of the two groups is 1:1, and total sample size = 200 cases of each of two groups calculated by PASS 15 software. Considering a loss to follow-up rate of 20%, a total sample size of 528 cases is required, and a sample size of 264 cases is required for each group. The sample size calculation formula is as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${n}_{T}={n}_{C}=\frac{{\left({Z}_{1-\alpha /2}+{Z}_{1-\beta }\right)}^{2}\left[{P}_{C}\left(1-{P}_{C}\right)+{P}_{T}\left(1-{P}_{T}\right)\right]}{{\left(\left|D\right|-\Delta \right)}^{2}}$$\end{document} n T = n C = Z 1 - α / 2 + Z 1 - β 2 P C 1 - P C + P T 1 - P T D - Δ 2 where P T and P C are the expected event rates of the test group and the control group, respectively; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left|D\right|$$\end{document} D is the absolute value of the difference between the expected rates of the two groups, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left|D\right|$$\end{document} D = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left|{P}_{T}-{P}_{C}\right|$$\end{document} P T - P C , and n represents the sample size of each group. Participants will be recruited from the outpatient departments of four tertiary hospitals in China, including Lanzhou University Second Hospital, the Third Affiliated Hospital of Wenzhou Medical University, Hebei Reproductive Health Hospital, and the First Affiliated Hospital of Chongqing Medical University. Women who present with a history suggestive of uRPL will be screened during routine consultations by trained trial staff. Eligible individuals will be provided with verbal and written information about the trial, and those who express interest will undergo further eligibility assessment and informed consent procedures. The random allocation sequence will be generated by the SAS9.2 statistical software package with a 1:1 allocation ratio. The random assignment sequence will be conducted and overseen by an investigator who will not be involved in participant recruitment, ensuring an impartial and unbiased sequence generation. The randomization sequence will be created prior to trial initiation and will be inaccessible to investigators, clinical staff, and participants to maintain blinding. Specifically, the random number table, the selected block length, and the random number initial value seed parameters are sealed in blinding codes as confidential data and stored in sequentially coded, sealed, and stamped radiopaque envelopes. The random allocation sequence will be generated by an independent statistician. Once a participant is deemed eligible and has provided written informed consent, the site investigator or designated study staff will access the system and enter the participant’s information. The system will then assign the participant to one of the two study arms according to the pre-generated randomization schedule. Allocation will be concealed from investigators, clinical staff, and participants throughout the trial.

Participants, care providers, investigators, outcome assessors, and data analysts will all be blinded to treatment allocation throughout the trial. Blinding will be achieved through the use of identically appearing rhG-CSF and placebo injections, which will be pre-packaged and labeled by an independent third party not involved in participant recruitment or outcome assessment. Unblinding will only be permissible in cases where knowledge of the participant’s treatment allocation is essential for clinical management, such as the occurrence of a serious adverse event that requires specific medical intervention. In such cases, the investigator must first consult with the coordinating center or sponsor to confirm the necessity of unblinding. If approved, unblinding will be performed using the secure randomization system, which allows authorized personnel to access allocation codes for individual participants. All instances of unblinding, including the reason, timing, and personnel involved, will be documented in the trial master file and reported to the ethics committee and sponsor as appropriate. Routine unblinding will not occur until the database is locked at the end of the study. Assessment and data collection will take place at the Department of Reproductive Medicine of Lanzhou University Second Hospital in Lanzhou, Department of Obstetrics and Gynecology of the Third Affiliated Hospital of Wenzhou Medical University in Wenzhou, Hebei Reproductive Health Hospital in Shijiazhuang, and the First Affiliated Hospital of Chongqing Medical University in Chongqing, with trained study staff conducting all procedures. To promote participant retention and ensure complete follow-up, study personnel will maintain regular contact with participants throughout the study period via phone calls, text messages, or clinic visits. Participants will be reminded of upcoming follow-up appointments and encouraged to attend even if they discontinue the intervention. Each center will assign a designated coordinator responsible for participant tracking and communication. If a participant withdraws from the intervention, efforts will be made to collect outcome data through scheduled visits or medical record review, unless the participant explicitly withdraws consent for follow-up. All study data will be collected using electronic case report forms hosted on a secure, password-protected, web-based electronic data capture (EDC) system. Data entry will be performed by designated staff at each study site and monitored by the coordinating center. The system will feature built-in range checks, validation rules, and logic checks to minimize data entry errors and ensure data quality. Data will be de-identified before entry, and all participants will be assigned unique study codes. Access to the EDC system will be restricted to authorized personnel with role-based permissions. Regular data monitoring will be conducted to identify inconsistencies, missing values, and protocol deviations, and queries will be issued to sites as needed. Final data cleaning and database locking will be performed prior to analysis. All data will be stored on encrypted servers with regular backups and will remain archived in accordance with national regulations and institutional policies. Biological samples will be handled under standard conditions with documented use or disposal and retained only for approved purposes with participant consent. Final data will be stored encrypted and may be shared post-publication upon request and approval. Each participant will be assigned a unique study identification code, and all clinical and laboratory data will be recorded in a de-identified format. Identifiable personal information (e.g., names, contact details) will be stored separately at each site in a secured, access-restricted file and will not be entered into the EDC system. Access to de-identified study data will be limited to authorized study personnel, and all data will be stored on encrypted servers with regular backups. Biological samples will be labeled using participant codes. Study findings will only be published in aggregated form, and no information that could identify individual participants will be disclosed. Biological specimens, including peripheral blood samples, may be collected from participants at baseline and/or during early pregnancy for exploratory immunological and molecular analyses related to uRPL. Samples will be processed at local laboratories according to standard operating procedures and then stored under appropriate conditions in coded, de-identified form. All specimens will be labeled with unique study identification codes, and any link to participant identity will be securely maintained at each study site. Sample use, transfer, and disposal will be documented, and all procedures will comply with national biobanking regulations and institutional policies. Our study team includes members with experience in clinical data analysis who will be responsible for conducting the planned statistical work. To further enhance methodological rigor and ensure proper handling of confounders, we plan to invite an experienced biostatistical expert to provide ongoing consultation and oversight throughout the data analysis phase. Statistical analysis will be performed using SAS software. All statistical tests are two-sided, and P  < 0.05 is considered as statistically significant for the tested difference (except where noted). u Quantitative variables will be described using mean, standard deviation, median, minimum, maximum, lower quartile (Q1), and upper quartile (Q3). Categorical variables will be described using number of cases and percentage of each category. u For the comparison of general conditions between the two groups, the appropriate method will be used for analysis according to the type of indicators; for the comparison of quantitative data between groups, the group t test (homogeneity of variance and normal distribution) or Wilcoxon rank sum test will be used according to the data distribution; for categorical data, chi-square test or exact probability method (if chi-square test is not applicable) will be used; for ranked data, Wilcoxon rank sum test or CMH test will be used. u Full analysis set (FAS) population: It refers to the subject set determined according to the principle of intention to treat. This data set is obtained after all the randomized subjects are eliminated in a minimum and reasonable method and contains all the randomized subjects who have received the investigational drug. FAS will be used for evaluation of primary and secondary indicators. u Per-protocol set (PPS) population: all patients complying with the trial protocol, with good compliance, and completing the treatment specified in the trial protocol without missing primary efficacy indicator. PPS set will be used for evaluation of primary and secondary indicators. u Safety set (SS): all the randomized patients who have received one dose of investigational drug and at least one safety assessment constitute the safety population in this study. The safety population is the primary population for safety evaluation in this study. Quantitative variables will be described using mean, standard deviation, median, minimum, maximum, lower quartile (Q1), and upper quartile (Q3). Categorical variables will be described using number of cases and percentage of each category. For the comparison of general conditions between the two groups, the appropriate method will be used for analysis according to the type of indicators; for the comparison of quantitative data between groups, the group t test (homogeneity of variance and normal distribution) or Wilcoxon rank sum test will be used according to the data distribution; for categorical data, chi-square test or exact probability method (if chi-square test is not applicable) will be used; for ranked data, Wilcoxon rank sum test or CMH test will be used. Full analysis set (FAS) population: It refers to the subject set determined according to the principle of intention to treat. This data set is obtained after all the randomized subjects are eliminated in a minimum and reasonable method and contains all the randomized subjects who have received the investigational drug. FAS will be used for evaluation of primary and secondary indicators. Per-protocol set (PPS) population: all patients complying with the trial protocol, with good compliance, and completing the treatment specified in the trial protocol without missing primary efficacy indicator. PPS set will be used for evaluation of primary and secondary indicators. Safety set (SS): all the randomized patients who have received one dose of investigational drug and at least one safety assessment constitute the safety population in this study. The safety population is the primary population for safety evaluation in this study. No interim analysis will be conducted. The number of enrolled cases and the number of completion cases in each site shall be summarized, and a list of dropout cases shall be provided. Different dataset sizes in each group, distribution of cases in each site, comparison of overall dropout rate and reasons for non-completion shall be listed in detail. Demographic characteristics (such as age and gender), relevant medical history, and treatment history of the patients are described, and the age, gender, and other characteristics of the two groups are compared to measure the comparability of the two groups. Based on the FAS and PPS, the difference in rates between the two groups and the 95% confidence interval are calculated. If the upper limit of the 95% CI of the difference in rates between the two groups is less than 0, the test group can be considered superior to the control group. The CMH chi-square test considering the site factor is used for comparison between groups. If it is site-adjusted, logistic regression model can be used for comparison between groups as appropriate. For secondary efficacy indicators, the corresponding statistical description and analysis methods will be selected based on the characteristics of indicators and general statistical principles. u Descriptive analysis will be performed for data of each safety indicator. All AEs occurring during the trial shall be recorded in detail. The number of occurrences, number of cases, and incidence of AEs shall be classified; summary and descriptive statistical analyses shall be performed, and a list shall be attached. AEs between the two groups are compared using the exact probability method. u Changes in laboratory parameters before and after treatment are described in cross-tabulations. Clinical judgment results (normal, abnormal without clinical significance, abnormal with clinical significance, and unchecked) of laboratory test results at each examination time before and after treatment are described by the number of cases and percentages using cross-tabulations. Safety indicators are based on the SS analysis set. Descriptive analysis will be performed for data of each safety indicator. All AEs occurring during the trial shall be recorded in detail. The number of occurrences, number of cases, and incidence of AEs shall be classified; summary and descriptive statistical analyses shall be performed, and a list shall be attached. AEs between the two groups are compared using the exact probability method. Changes in laboratory parameters before and after treatment are described in cross-tabulations. Clinical judgment results (normal, abnormal without clinical significance, abnormal with clinical significance, and unchecked) of laboratory test results at each examination time before and after treatment are described by the number of cases and percentages using cross-tabulations. Safety indicators are based on the SS analysis set. Subgroup analysis will be performed based on pre-specified grouping: research centers (4 centers), age, number of pregnancy losses (2 or 3), and BMI. The reasons for withdrawal from both randomization groups will be documented and qualitatively compared. The impact of missing data on study outcomes will be evaluated through sensitivity analyses (worst case and best case) using augmented datasets. To address missing baseline data, the multiple imputation method will be applied, assuming that the data are missing at random. Regarding secondary outcomes, although they are primarily exploratory, we acknowledge the risk of inflated type I error due to multiple comparisons. Therefore, we will consider applying appropriate multiple testing correction methods (e.g., Bonferroni or Benjamini–Hochberg procedures) to enhance the interpretability and reliability of the results.

URPL poses a significant emotional and physical burden on affected couples, yet there remains a lack of both effective and safe treatment strategies. Among the many proposed interventions, G-CSF has emerged as a promising candidate due to its immunomodulatory properties and role in promoting implantation and placental development [ 29 ]. G-CSF, a hematopoietic growth factor primarily used for treating neutropenia resulting from various factors, has been found to reduce rates of pregnancy loss, with evidence mostly derived from the Severe Chronic Neutropenia International Registry [ 30 ]. Additionally, G-CSF plays a crucial role in regulating normal pregnancies, promoting maternal–fetal immune tolerance, enhancing endometrial receptivity, and facilitating vascular remodeling at the maternal–fetal interface. G-CSF can induce the transformation of Th1 to Th2 cells, increase Treg counts in the body, and decrease NK cells in the peripheral blood of pregnant women. This maintains maternal–fetal immune balance and plays a critical role in embryo implantation and sustaining a healthy pregnancy [ 29 ]. These mechanisms provide biological plausibility for its use in patients with presumed immunological causes of pregnancy loss. Several studies have demonstrated the potential benefit of G-CSF treatment for uRPL, but the conclusions drawn from these studies have been disputed. Scarpellini et al. [ 16 ]reported a significantly improved live birth rate with G-CSF compared to placebo, while Eapen et al. [ 17 ]did not replicate this benefit in a larger multicenter trial. A recent meta-analysis further suggested that G-CSF may significantly improve the conception rate (RR = 1.34, 95%CI: 1.03–1.74) but does not clearly increase live birth rates (RR = 1.35, 95%CI: 0.99–1.84), highlighting the need for high-quality, adequately powered trials to clarify its therapeutic role [ 18 ]. As far as we know, existing RCTs investigating the use of G-CSF for uRPL patients have included mostly limited sample sizes. Furthermore, these RCTs display variability in terms of patient inclusion criteria, routes of administration, time of medication initiation, duration of treatment, and dosages [ 16 ], [ 17 ], [ 31 ],[ 32 ] [ 33 ]. Therefore, the therapeutic efficacy of G-CSF in patients with uRPL necessitates high-quality, large-sample size, double-blind RCTs to verify its effectiveness and safety. In turn, the optimal administration timing, dosage, duration, and route should be established, alongside clear indications and contraindications, thus providing better clinical applications of G-CSF in the uRPL field. This protocol outlines a large-scale, multi-center, double-blind RCT designed to evaluate the efficacy and safety of G-CSF in Chinese women with uRPL. The study population is rigorously selected according to our inclusion and exclusion criteria. Enrolled participants will be randomly assigned into either the test group or control group on the day of the positive pregnancy test. Compared to the sample size involved in previous RCTs, this trial has a significant upgrade, with an estimated sample size of 264 participants per group. Several studies have also provided evidence on the safety of G-CSF administration during pregnancy. In a randomized controlled trial, Scarpellini et al. [ 16 ] reported no significant differences in neonatal weight, congenital anomalies, or perinatal complications between the G-CSF group and the placebo group. Likewise, a multicenter randomized clinical trial conducted by Eapen et al. [ 17 ] found that G-CSF treatment did not increase the incidence of adverse events in newborns. These findings support the overall fetal and neonatal safety profile of G-CSF when used during early pregnancy, although long-term postnatal outcomes still require further research. Although the present study is ongoing and remains blinded, trial staff have observed several cases in which RPL women have successfully progressed beyond 24 weeks’ gestation. While these observations cannot be interpreted as formal results, they offer encouraging signals and reinforce the value of continuing the trial. Final conclusions regarding efficacy and safety will only be drawn after the full dataset has been unblinded and analyzed according to the predefined statistical plan. Despite these strengths, the study has several limitations. First, as the trial is conducted exclusively among Chinese women, the generalizability of findings to other ethnic groups or healthcare settings may be limited. Second, due to feasibility and resource constraints, certain potential etiological factors, such as paternal chromosomal anomalies or sperm DNA fragmentation, were not assessed at baseline, and thus their influence cannot be excluded. Third, although short-term neonatal outcomes will be collected, long-term follow-up of infants is not planned in this protocol, and potential late-onset adverse effects may be missed. Finally, recruitment may face challenges due to the strict eligibility criteria and requirement for early pregnancy identification. In summary, this study aims to generate high-quality evidence on the efficacy and safety of G-CSF in uRPL. If successful, it may lead to changes in clinical practice and offer a viable treatment option for women affected by this distressing condition; this would signify a significant change in treatment policies and associated guidelines, and potentially serve as a reliable and safe therapy approach in the future. Until the results become available, caution remains warranted in the routine clinical application of G-CSF for uRPL. Four centers are currently actively enrolling participants. Recruitment began on November 9, 2023. The trial is expected to be completed by April 30, 2025. The trial, first version, was registered on November 8, 2023.

Recurrent pregnancy loss (RPL), defined as the loss of two or more pregnancies, affects approximately 1–2% of women attempting to conceive [ 1 ]. Potential contributors to RPL include genetic abnormalities, immunological dysfunction, uterine structural anomalies, endocrine disorders, and thrombophilia, although more than 50% of cases remain unexplained [ 2 ]. Clinically, uRPL poses a serious negative impact on the physical and mental health, particularly on women. Anxiety, depression, guilt, and relationship stress often worsen with each loss, severely affecting the quality of life [ 3 ]. Growing evidence suggests that immune dysregulation at the maternal–fetal interface plays a key role in unexplained RPL (uRPL) [ 4 ]. Maternal rejection of the embryo (semi-allograft) may be an important causative factor of pregnancy loss [ 5 ]. Multiple immunotherapies have been tried in clinical practice, including lymphocyte immunization therapy (LIT), glucocorticoids, intravenous injection of immunoglobulin (IVIG), intravenous injection of fat emulsion, and anti-tumor necrosis factor-α monoclonal antibody therapy [ 6 ] [ 7 ] [ 8 ]. However, their efficacy remains controversial [ 9 ]. A systematic review of 20 high-quality studies found that regimens such as LIT and IVIG failed to significantly improve pregnancy outcomes in uRPL, highlighting the need for new therapeutic options [ 10 ]. Granulocyte colony-stimulating factor (G-CSF), a glycoprotein growth factor commonly used for neutrophils, has recently attracted interest in reproductive medicine [ 11 ],[ 12 ]. Beyond its hematologic effects, G-CSF has been shown to promote trophoblast proliferation and invasion, shift Th1/Th2 balance toward tolerance, and modulate regulatory T-cell activity, mechanisms potentially beneficial in uRPL [ 13 ],[ 14 ],[ 15 ]. However, clinical trials have yielded conflicting results. Scarpellini et al. conducted a 7-year randomized controlled trial (RCT) in 68 patients with uRPL and reported a significantly higher live birth rate in the G-CSF group compared to the placebo group (82.8% vs 48.5%) [ 16 ]. In contrast, a larger multicenter randomized double-blind controlled trial by Eapen et al. did not demonstrate a significant difference in the 20-week live birth rate (59.2% vs 64.9%) [ 17 ]. A recent meta-analysis concluded that while G-CSF may improve conception rates, its impact on live birth remains inconclusive [ 18 ]. These inconsistent findings underscore the need for further high-quality research to evaluate the efficacy of G-CSF in uRPL, particularly in the Chinese population. Given potential genetic, environmental, and clinical practice differences, it is essential to generate high-quality evidence in the Chinese population, where data on G-CSF use in uRPL remain limited. Emerging evidence suggests that microRNA polymorphisms, which influence immune regulation and endometrial receptivity, may differ across ethnic groups [ 19 ],[ 20 ][ 21 ]. Domestic studies on G-CSF in the treatment of uRPL are mostly limited by sample size and methodological shortcomings, and their outcome indicators vary greatly [ 22 ], [ 23 ]. In summary, it is not determined yet whether G-CSF can be used to treat uRPL. Therefore, it is urgently necessary for domestic research evidence with high quality to validate the efficacy of G-CSF in Chinese patients with uRPL, so as to provide more theoretical basis for clinical practice. The present study aims to design a multicenter, double-blind RCT to evaluate the efficacy and safety of G-CSF in Chinese women with uRPL. We hypothesize that G-CSF improves pregnancy outcomes by modulating maternal–fetal immune tolerance and aim to assess whether it can improve fetal survival in this population. A randomized, double-blind, placebo-controlled, multicenter clinical trial with a 1:1 allocation ratio to either G-CSF or placebo arms will be conducted, adhering to the SPIRIT reporting guidelines (Figs. 1 and 2 ). The trial is being conducted across four reproductive centers in China and will evaluate the efficacy and safety of recombinant human G-CSF in Chinese women with uRPL. The flowchart was in accordance with Standard Protocol Items: Fig.  1 provides a graphical summary of the trial design, including participant recruitment, allocation, therapy, and follow-up. Fig. 1 Flowchart for enrollment, allocation, treatment, and follow-up of study participants Fig. 2 SPIRIT figure reporting the phases of the trial and data collection time point Flowchart for enrollment, allocation, treatment, and follow-up of study participants SPIRIT figure reporting the phases of the trial and data collection time point In the development of the study protocol, there was no direct involvement from the public or patients, but it was designed based on current clinical evidence and expert consensus in reproductive medicine.